Marine Biology (2018) 165:19 https://doi.org/10.1007/s00227-017-3274-y

ORIGINAL PAPER

A synthesis of European seahorse taxonomy, population structure, and habitat use as a basis for assessment, monitoring and conservation

Lucy C. Woodall1,2 · Francisco Otero‑Ferrer3 · Miguel Correia2,4 · Janelle M. R. Curtis5 · Neil Garrick‑Maidment6 · Paul W. Shaw7 · Heather J. Koldewey2,8

Received: 15 August 2017 / Accepted: 22 November 2017 © The Author(s) 2017. This article is an open access publication

Abstract Accurate taxonomy, population demography, and habitat descriptors inform species threat assessments and the design of efective conservation measures. Here we combine published studies with new genetic, morphological and habitat data that were collected from seahorse populations located along the European and North African coastlines to help inform manage- ment decisions for European seahorses. This study confrms the presence of only two native seahorse species (Hippocampus guttulatus and H. hippocampus) across Europe, with sporadic occurrence of non-native seahorse species in European waters. For the two native species, our fndings demonstrate that highly variable morphological characteristics, such as size and pres- ence or number of cirri, are unreliable for distinguishing species. Both species exhibit sex dimorphism with females being signifcantly larger. Across its range, H. guttulatus were larger and found at higher densities in cooler waters, and individuals in the Black Sea were signifcantly smaller than in other populations. H. hippocampus were signifcantly larger in Senegal. Hippocampus guttulatus tends to have higher density populations than H. hippocampus when they occur sympatrically. Although these species are often associated with seagrass beds, data show both species inhabit a wide variety of shallow habitats and use a mixture of holdfasts. We suggest an international mosaic of protected areas focused on multiple habitat types as the frst step to successful assessment, monitoring and conservation management of these Data Defcient species.

Introduction

The paucity of species-specifc data is among the many chal- lenges to designing efective marine conservation measures Responsible Editor: K. D. Clements. that are resilient to the enduring threats of climate change, coastal development, over-fshing, by-catch efects and inva- Reviewed by R. Calado and an undisclosed expert. sive species (Klein et al. 2013; Selig et al. 2014). These challenges are further compounded when the taxonomy Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00227-017-3274-y) contains of species is uncertain. Knowing which species occur and supplementary material, which is available to authorized users.

* Lucy C. Woodall 5 Pacifc Biological Station, Fisheries and Oceans Canada, [email protected] 3190 Hammond Bay Road, Nanaimo, BC, Canada 6 The Seahorse Trust, 36 Greatwood Terrace, Topsham, 1 Department of Zoology, University of Oxford, Oxford, UK Devon, UK 2 Project Seahorse, Zoological Society of London, Regent’s 7 Institute of Biological, Environmental and Rural Sciences Park, London, UK (IBERS), Aberystwyth University, Aberystwyth, UK 3 Grupo en Biodiversidad y Conservación, IU-ECOAQUA, 8 Centre for Ecology and Conservation, University of Exeter, Universidad de Las Palmas de Gran Canaria, Crta. Taliarte Penryn, UK s/n, 35214 Telde, Spain 4 CCMar, Universidade do Algarve, F. C. T., Edifcio 7, Campus de Gambelas, 8005‑139 Faro, Portugal

Vol.:(0123456789)1 3 19 Page 2 of 19 Marine Biology (2018) 165:19 understanding their life-history, ecology, and behaviour is currently targeted by fsheries throughout most of their geo- increasingly important to ensure efective and robust con- graphic range, but there is trade in west Africa of H. hip- servation and management (Perry et al. 2005; Lavergne et al. pocampus (Cisneros-Montemayor et al. 2016) and a new and 2010; Dawson et al. 2011). increasing fshery for H. guttulatus in the Ria Formosa in The cryptic nature of seahorses (genus Hippocampus) has Portugal (M. Correia, pers. obs.). Both species are also sus- led to signifcant confusion regarding their taxonomy and ceptible to anthropogenic activities and habitat loss (Curtis ecology, which poses challenges to managing the activities et al. 2007). Ecological data on seahorses are scarce due to that threaten these fshes. The most recent and comprehen- their apparent patchy distribution and low density, as well sive taxonomic review suggests there are two native species as their cryptic nature (Foster and Vincent 2004). These fea- of seahorse in European waters, H. guttulatus and H. hip- tures make them particularly difcult to survey, assess and pocampus (Lourie et al. 2016), but considerable intraspecifc monitor the status of their populations, either for scientifc variability in morphology within this genus (Lourie et al. research or commercial development projects, such as envi- 1999b; Otero-Ferrer et al. 2017) has led to much confusion ronmental impact assessments prior to construction work. regarding their taxonomy, and the taxonomy and nomen- To date, a range-wide ecological assessment has been clature of these species is not stable. Authors previously conducted for just one seahorse species (H. capensis), which suggested many additional species within this geographic is confned to three estuaries in South Africa (Lockyear et al. range, based on small morphometric diferences (Kuiter 2006). For European seahorses, research has generally been 2009). For instance a study by Vasil’Eva (2007), which has limited to small focal sites (e.g. Curtis and Vincent 2006; not been adopted (Eschmeyer and Fricke 2016), attempted to Gristina et al. 2015) or collection of qualitative data (e.g. change the names of these species, while another author sug- Filiz and Taskavak 2012). However, a very large sighting gested additional species were present based on photographs dataset has been collected for UK and Ireland (N. Garrick- (Kuiter 2009). There is ongoing discussion as to whether Maidment pers. comm.). Comparisons of population struc- H. ramulosus is a simple synonym of H. guttulatus, and ture among studies is also challenging because seahorse whether the regional morphological diferences observed length can be measured by standard length (LS), total length across the seahorse populations in the region are indica- (LT) or height (Lourie et al. 1999a) and previous studies have tive of diferent species (Kuiter 2009). Taxonomic contro- used all of these (e.g. Verdiell-Cubedo et al. 2008; Nadeau versy involving splitting and lumping of species is common et al. 2009; Caldwell and Vincent 2012; Vieira et al. 2014). throughout the Syngnathidae family, due to limited discrimi- Focusing on the taxonomy, biology and life history of nating morphological characteristics between species and European seahorses, we use published and unpublished the ability within the family to change colour and cirri (fla- sources of genetic, demographic and environmental data to mentous skin appendages) (Curtis 2006). As most ecological investigate the following objectives: studies of seahorses in Europe have used the nomenclature of H. hippocampus and H. guttulatus to defne their focal 1. Use genetic markers to confrm the number of seahorse species (e.g. Curtis and Vincent 2005; Kitsos et al. 2008; species present in Europe Ben Amor et al. 2011; Caldwell and Vincent 2012; Filiz 2. Test for diferences in population structure and behav- and Taskavak 2012; Gristina et al. 2015), there is some con- iour throughout the range sensus for a conservative view of seahorse taxonomy. Some 3. Test for the correlation of population structure and mor- reports also suggest range extensions into European waters phology with environmental variables by non-native species: H. algiricus presence in the Canary Islands (Otero-Ferrer et al. 2015b, 2017), the Lessepsian This information will help to advance our ability to efec- migrant H. fuscus in the eastern Mediterranean (Golani and tively manage Hippocampus spp. within Europe, by provid- Fine 2002), and occasional rare migrants (e.g. H. erectus, ing detailed information that can help determine appropriate Woodall et al. 2009). Therefore genetic data are particularly protection and mitigation interventions as well as the accu- useful to clarify taxonomy and complement morphological rate assessment of seahorse populations. data (Padial et al. 2010). The two European seahorses H. guttulatus and H. hip- pocampus are the currently recognised names used in the Materials and methods IUCN Red List of Threatened Species (2012) and both are currently assessed as Data Defcient (Woodall 2012a, b). Geographic extent, literature sources Both species have a large geographic range extending across and standardization most of Europe and North Africa including the Atlantic Ocean, Mediterranean and Black Seas (Lourie et al. 1999b; The geographic extent of this review covers seahorse pop- Otero-Ferrer et al. 2017). Neither species is thought to be ulations from the Northeast Atlantic Ocean, including the

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Macaronesian islands, and the Mediterranean and Black New sample acquisition and genetic analysis Seas. In total, data from 13 countries and 37 diferent sites are reviewed. These data cover the known geographic Most specimen tissue was collected during sampling dives. range of H. guttulatus and H. hippocampus (Lourie et al. Further specimens or tissue and associated data were also 1999b), however most individual studies generally focused donated by fshers, public aquariums and academics, and on sites in Portugal and the eastern Mediterranean due to were used when source location was known. In total, sea- these being identifed as having relatively high seahorse horse tissue was obtained from specimens from 18 sites abundance that led to longer term studies. Data used in around Europe and North Africa (Fig. 1). Authors directly this review were from a wide range of sources, including sampled tissues from 14 sites, while tissue from the remain- sources known to authors or found using a combination ing four locations was donated by other researchers. The of the following search terms ‘Seahorse, Hippocampus, mitochondrial DNA cytochrome b gene (cytb) and Control guttulatus, ramulosus, Mediterranean, Atlantic, Black Sea, Region (CR) were amplifed from specimens using methods short snouted, long snouted, Hippocampe, Caballito de given in Woodall et al. (2011, 2015). All DNA sequences Mar, cavalos marinhos’ in search engines Google Scholar were deposited in Genbank (Table S1). Cytb is most rou- and Web of Science. Sources include new data and pub- tinely sequenced in seahorses, and thus provided an oppor- lished literature comprising peer-reviewed papers, theses, tunity to include the greatest number of species. The cytb books and grey literature such as conference posters and sequences were combined with seahorse reference sequences reports. Due to the diversity of the methods employed by (Casey et al. 2004; Teske et al. 2007a, b), and aligned using these studies, we were not able to use all data from all ClustalW (Larkin et al. 2007) implemented in Geneious studies in comparisons among sites. However, all den- v6.1.7. The pipefsh Syngnathus temminckii was used as an sity measures were standardised to ind. m­ −2 and seahorse outgroup. A phylogenetic tree, to group similar haplotypes, length measurements were compiled as standard length was created in Mr. Bayes (Huelsenbeck and Ronquist 2001) (LS) (Curtis and Vincent 2006), height (Foster and Vincent implemented in Geneious after using Find model (Posada 2004) or total length (LT) (Verdiell-Cubedo et al. 2008). and Crandall 2001) to determine the most suitable nucleo- tide substitution model (GTR + γ) for this dataset.

Fig. 1 Locations of seahorse tissue collection, population demography and environmental data, including site codes. Filled shapes are sites with new data and open shapes are sites with published data

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Environmental and demographic data collection located were recorded, but using this search pattern it was not possible to record the area of benthos searched. Environmental and demographic data were collected oppor- Search efort was measured by time and the number of tunistically during SCUBA dives, and morphological data searching divers, so the relative abundance of seahorses from donated specimens as detailed below. Ideally, a strati- (seahorse per diver hour) could be reported (Schmitt fed random or systematic sampling regime would be used and Sullivan 1996). to capture the full range of diversity in genetic and demo- (b) Transect dives graphic structure and to identify environmental correlates. A random position within the general search area was However, this was not feasible at the geographic scale of assigned as the starting point. This point was defned interest because so little information is known of seahorse either by its GPS position or by its bearing and dis- distributions, which are generally patchy and low-density tance from a known structure (e.g. pier or rocky fea- (Curtis and Vincent 2005). Instead local knowledge of sea- ture). From the starting position, a 30 m tape was laid horse occurrences was used to identify suitable and accessi- out by one diver in a random direction while the other ble survey locations. All seahorses encountered were identi- diver recorded the number of seahorses by species, sex fed in the feld as either H. guttulatus or H. hippocampus, and holdfast within a 2 m corridor belt transect centred using morphological characteristics that have proved to along the tape length. Returning along the transect, be robust such as head shape and head:snout ratio (Lourie both divers assessed the habitat by determining the et al. 1999b; Curtis 2006). Photographs of specimens were dominant habitat type, which was defned by the broad taken when possible and representative images displayed in categories given above. The divers also determined the Fig. S1. Water temperature was extracted from http://www. percentage of cover of each habitat type within three seatemperature.org (1st July 2015) and was used for all sites randomly positioned 1 m2 quadrats. This process was where published studies were conducted. repeated so that a total of four transects were surveyed per site. At one site in Greece (KGR—Fig. 1), the tran- Survey data collected by divers sects originated from a start line running at right angles to the slope. These transects were positioned to run At 14 of the 37 sites, diving methods were employed to sam- parallel to the slope contours, at randomly assigned dis- ple seahorse populations in two ways: (a) to collect tissue tances along the starting line. The deepest transect was samples for genetic studies (collection dives—see Woodall surveyed frst and the shallowest last. This method was et al. 2011); or (b) to carry out rapid population assessments necessary at KGR as it had a rapidly sloping benthic (transect dives—adapted from Curtis and Vincent 2005; profle, which was absent from other sites. Woodall et al. 2015). All dives were conducted during the main breeding season for the seahorses (May–October, Cur- Commercial trade and fshing data tis and Vincent 2006). During both dive types (collection and transect), once individual seahorses were located, their Fisher data were collected from two locations in the UK. holdfast (seagrass species, algae species, artifcial structures, The seahorses were accidentally captured in gill nets and sand, shells, sessile invertebrates), depth and macro habitat crab pots by local fshers who were targeting Solea solea (seagrass, macroalgae, sessile invertebrates, sponge, sand/ and Cancer pagurus. Undamaged seahorses were returned mud, stones/pebbles, rocks, clif or artifcial structures) were to the water and injured ones were donated to local public recorded, as well as species, sex, maturity (size when brood aquariums. Environmental (habitat and depth) and seahorse- pouch is mature in males of that population; Curtis and Vin- specifc data (species and number) were recorded on the fsh- cent 2006; Curtis et al. 2017), presence or absence of cirri ing boat and at the aquariums. Seahorses were donated by (Curtis 2006) and straight trunk length (LTr) which was later researchers from three locations in France, Portugal and Italy converted to standard length (LS) (Curtis and Vincent 2006). (AFR, TPO and RIT Table 1, Fig. 1). Seahorses from site Water clarity was assessed during each dive by estimating AFR were collected during experimental trawls that were the horizontal visible distance (meters). The mean tempera- used to survey fsh diversity in the bay. These seahorses were ture at each site was calculated and recorded using a dive returned to their collection site following LTr measurement computer (Mosquito, Suunto). and photographing. Sites TPO and RIT were fshed using beach seine nets by aquarium staf for specimen provision (a) Collection dives to local public aquariums. Specimens were measured and Between two and five (site dependant) divers photographed by aquarium staf. Specimens from Senegal searched the benthic substratum for seahorses using (SEN) were donated by Project Seahorse. Most of these a random search pattern. The total time spent search- Senegalese samples were obtained from a traditional medi- ing (diver hours) and number of individual seahorses cine market (Hong Kong) by representatives of a Project

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Table 1 Sample sites of seahorses Hippocampus guttulatus (G) and Transect), Social (Donations, Interviews or Trade)], and environmen- H. hippocampus (H), sampling method [Fishing Method (Net, Pot, tal parameters (depth, visibility, temperature, habitat and main sea- Trawl, Trammel or Dredge), Type of dive (Collection, Survey or horse holdfast), (a) new data (b) published data (a) New data Site Country Location type Species Sampling Depth range (m) Visibility (m) Tem- Holdfast Habitat method perature (°C)

HUK UK Coast H Fisher–Nets ≈ 55 n/a n/a Plocamium spp. Sand and mac- roalgae SUK UK Coast H Fisher–Pots ≈ 25 n/a n/a n/a Mussel bed BFR France Coastal H, G Collection dive 2–6 16 Z. marina, Ulva Z. marina beds spp., Sabel- lidae spp. AFR France Lagoon G Donation 5–10 n/a n/a n/a Channel of sand with Z. marina beds on sides TPO Portugal Lagoon G Donation, col- 3–4 2–4 19 Z. marina Z. marina beds on lection dive sand PPO Portugal Estuary G Collection dive 1–3 < 1 19 Artifcial Ropes and other artifcial struc- tures on heavy silt RPO Portugal Lagoon H, G Collection dive 1–6 1–7 20 Sand, Z. marina, Sparse Z. marina, C. nodosa, sand urchins and Artifcial macro-algae, tunicates and artifcial MSP Spain Coastal H, G Collection dive 6–8 2–5 19 Z. marina Mixed sparse seagrass beds TFR France Lagoon G Collection dive 2–4 1–4 21 Various Mixed and com- plex GFR France Coastal H Collection dive 4.5–6 < 1 15 On benthos Heavy silt and tunicates GMA Malta Coastal H Collection dive 9–20 15–40 18 Z. marina Sand/seagrass bed, + 70 m deep wall @ 20 m KGR Greece Coastal H, G Collection dive 5–19 15–20 24 Z. marina Mixed seagrass on transect dive slope CGR​ Greece Coastal G Collection dive 2–5 15–20 26 Stones Sponge, rock and transect dive pebble wells VBU Bulgaria Coastal G Collection dive 5–6.5 1 25 In mixed algae, Ulva spp. transect dive Dictyopteris and Chaeto- morpha spp. LCN Spain Coastal H Collection dive 6–21 10–30 21 Artifcial sub- Rock, rope and transect dive strates ship wreck SEN Senegal n/a H Trade n/a n/a n/a n/a n/a (b) Published data Site Country Type Species Sampling method Depth range (m) Holdfast Habitat References

DUK UK Coastal G Survey dive 1–3 Z. marina Z. marina Garrick-Maidment et al. (2010) OUK UK Lagoon H, G Interview 0–17 Algae and seagrass Algae (H + G), N. Garrick-Maidment (G) n/a (H) sand (H + G), Pers. Corr. mixed seagrass (G), Oyster bed (H + G)

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Table 1 (continued) (b) Published data Site Country Type Species Sampling method Depth range (m) Holdfast Habitat References

AFR France Lagoon H, G Interview 3–20 n/a Z. marina, Z nolti, Grima (2011) sand, shells GSP Spain Coastal H, G Survey dive 2.5–8 Macroalgae (G) Sand (G), seagrass Valladares et al. Seagrass (H) macroalgae (2011), (2013) APO Portugal Estuary H, G Net n/a n/a n/a Veiga et al. (2009) RPO Portugal Lagoon H, G Transect dive and 0–7 Tunicates and shells Mixed seagrass and Curtis and Vincent net (H + G), Artifcial macroalgae (G), (2005, 2006), Curtis (G), sessile inver- sessile inverte- (2004), Caldwell tebrates (H + G), brates (H + G), and Vincent (2012), macroalgae sand (H) Correia et al. (H + G), seagrass (2015), Vieira et al. (H + G) (2014) RSP Spain Lagoon G Net 2–3 n/a C. nodosa, invasive Verdiell-Cubedo Caulerpa et al. (2006) TFR France Lagoon G Transect dive 0–9 Artifcial Sand, algae and Louisy (2011) sparse seagrass SIT Italy Coastal H Transect dive n/a n/a Sand Canese et al. (2007) MIT Italy Lagoon H, G Transect dive 0–5 12 Artifcial (H + G) Mixed sand, sparse Tiralongo and Balda- seagrass, dense cconi (2014), Gris- Ulva sp. tina et al. (2015) VIT Italy Lagoon H, G Net n/a n/a Seagrass (H + G), Franco et al. (2006) saltmarsh (G) TSL Slovenia Coastal G Transect dive 4–10 C. nodosa C. nodosa Bonaca and Lipej (2005) KGR Greece Coastal G Transect dive n/a n/a n/a Issaris and Katsane- vakis (2010) MTR Turkey Coastal G Net 0–2 n/a Seagrass, sand Keskin (2007) TTR​ Turkey Coastal G Interview n/a n/a n/a Kasapoglu and Duz- gunes (2014) NTR Turkey Coastal G Interview n/a n/a n/a Başusta et al. (2014) WTR​ Turkey Coastal H, G Interview 0–30 n/a Seagrass, rock, Filiz and Taskavak mud, sand (2012) ATR​ Turkey Coastal H, G Interview and trawl n/a n/a n/a Gurkan and Taskavak (2007) RGR​ Greece Coastal H, G Trawl 12–15 n/a seagrass Kitsos et al. (2008) GTU​ Tunisia Lagoons H, G Trammel net and n/a n/a n/a Ben Amor et al. dredge gear (2011) GCN Spain Coastal H Transect dive 15 Macroalgae, sessile Rock, sand, C. Otero-Ferrer (2011) invertebrates, nodosa, macroal- seagrass gae, artifcial

Seahorse/TRAFFIC partnership, but twelve were obtained or a combination of the two. A post hoc Tukey pairwise by K. West directly from Senegal traders (West 2012). comparison was used to determine at which sites seahorse lengths were signifcantly diferent. Diferences in seahorse Statistical analysis abundance were determined by Mann–Whitney test, and cor- relation between abundance of the two species was assessed Pearson correlation was used to assess all correlation rela- with Pearson correlation. Deviation of sex ratios from equal tionships between seahorse size, sex and cirri presence. was measured with Chi squared goodness of ft, and post Diferences in seahorse length, once juvenile data (Cur- hoc multiple test Benjamini–Hochberg correction. Correla- tis and Vincent 2005) were removed, were assessed using tion between abiotic parameters and species abundance was GLM to determine whether there was a diference between assessed with Spearman Rho. All these tests were imple- sexes and between sites (when mature seahorse n > 10), mented in Minitab v 17. Association of species presence

1 3 Marine Biology (2018) 165:19 Page 7 of 19 19 with specifc habitat parameters was calculated by ANOSIM specimens of H. fuscus from Egypt, and fve specimens of in Primer v7. H. algiricus from Senegal (cytb data only shown, Fig. 2). All other specimens clearly group into two monophyletic clades corresponding to the two recognised European Results species H. guttulatus (212) or H. hippocampus (257). Intraspecifc DNA sequence variation across all samples Genetic diferentiation of H. guttulatus and H. hippocampus was low (1.23% cytb and 1.49% CR, and 1.94% cytb and 1.96% CR, respec- In total, 478 seahorse specimens from 18 sites represent- tively), and identical (H. guttulatus) or similar (H. hip- ing 10 countries (Fig. 1, Table 1) were PCR amplifed and pocampus) to variation observed within individual popula- sequenced for both cytb and CR regions, with fragments tions [maximum of 1.23% for cytb (VBU) and 1.49% for trimmed to 518 and 397 bp, respectively to assist align- CR (MSP) in H. guttulatus, and 1.21% for cytb (RPO) and ment. Data suggested the presence of a single specimen 1.67% for CR (SEN) in H. hippocampus—see Woodall of H. erectus from the Azores (Woodall et al. 2009), three et al. (2011, 2015)].

Fig. 2 Phylogenetic tree of the relationship among Hippocampus species, constructed from Cytochrome b using MrBayes (GTR + γ) and shows posterior probability. Shaded labels are those generated in this study, and H. hippocampus from Senegal are denoted by bold text

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Seahorse morphology sexes, sites and the interaction of the two, accounting for 55% of the variation seen (GLM, sex F = 50.8, P < 0.001, Cirri were present on both species and the number of cirri sites F = 4.9 P < 0.005, sex and site F = 8.6, P < 0.001). present on seahorses varied considerably between sites and Similar to H. guttulatus, male H. hippocampus were signif- between individuals within sites. The mean cirri presence cantly smaller than the females according to Tukey’s pair- for H. guttulatus was 87% (40–100%, N = 500) across 11 wise comparisons at 95% CI, and individuals from Senegal sites and for H. hippocampus the mean was 43% (6–74%, were signifcantly larger than those of all other sites (Fig. 3, N = 226) across 9 sites, covering the entire geographic range Table 2). In both species there is no correlation between lati- of these species. For both species the number of cirri varied tude and standard length (H. guttulatus Pearson correlation, more between individuals within the same site than between R = 0.23, N = 15, P = 0.42; H. hippocampus R = − 0.362, species; however the species difered signifcantly in num- N = 16, P = 0.17). ber of cirri (Mann–Whitney, W = 157, N1 = 11, N2 = 98, P < 0.005). In H. guttulatus, data from this study show there Seahorse population density is no correlation between sex and cirri presence (Pearson correlation, R = − 0.11, N = 151, P = 0.16), however stand- The total number of seahorses observed at each site var- ard length and cirri presence are signifcantly correlated with ied considerably depending on species and survey method. larger fsh having cirri more often than smaller fsh (Pearson The mean abundance of H. guttulatus was 3.15 ± 1.08 correlation, R = 0.40, N = 151, P < 0.001). By contrast (mean ± SE) seahorses per diver hour for collection dives, female H. hippocampus were more often seen with cirri than and in transect surveys density was 0.076 ± 0.06 ind. ­m−2. males (Pearson correlation, R = − 0.27, N = 91, P < 0.01). For H. hippocampus, the mean abundance was 2.49 sea- Standard length of H. guttulatus was signifcantly difer- horses per diver hour but only one transect was conducted ent between sexes and between sites (GLM, sex F = 39.4, for this species, so no mean density is reported. When data P < 0.001, sites F = 47.7, P < 0.001, site and sex F = 0.47, from published studies were combined with transect dives P = 0.83). Males were signifcantly smaller than females, in this study, mean abundance was greater in H. guttulatus and H. guttulatus in the Black Sea were signifcantly smaller than H. hippocampus (H. guttulatus: 0.04 ± 0.01 ind. m­ −2 than at any other site according to Tukey’s pairwise com- N = 12; H. hippocampus: 0.003 ± 0.001 ind. ­m−2, N = 7) parisons at 95% CI. When new and published data are com- (Table 3). bined, individuals of H. guttulatus in the Black Sea (VBU In all locations (N = 6) where the two species occurred and TTR, Fig. 1) are smaller than specimens sampled from sympatrically, the density of H. guttulatus was greater than everywhere else (Fig. 3, Table 2). In H. hippocampus there that of H. hippocampus and in all but one case this was by were significant differences in standard length between at least an order of magnitude greater (Table 3). In the Ria

Fig. 3 Standard length of H. guttulatus (squares) and H. hippocampus (circles) includ- ing new data and that from published studies, when n > 10. Filled in shapes are new data and open shapes are data from previous studies (Table 1 for site code details). Sites are grouped by region and are ordered from north to south or west to east depending on location

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Table 2 Population demographics of Hippocampus guttulatus (a) and H. hippocampus (b)

Site Total number Juvenile (%) Proportion of Ls (cm) Sampling period References females (min and max)

(a) Hippocampus guttulatus PUK 17 0 0.53 13.6 (8.6–18.6) May, Aug, Oct This study OUK 28 n/a n/a 15.7 (10.0–21.6)b n/a Neil Garrick-Maidment Pers. Corr. BFR 15 1 0.60 15.2 (8.0–20.5) June This study AFR 38 3 0.71 13.0 (8.6–17.4) Sept, Oct, Nov This study GSP 21 14 0.33 n/a Year-round Valladares et al. (2011) APO 84 n/a n/a 12.8 (3.6–18.5) Year-round Veiga et al. (2009) TPO 37 8 0.50 13.2 (9.0–20.4) Sept, Oct This study PPO 42 43 0.45 13.9 (9.0–16.8) Sept This study RPO 321 17 0.57 12.7 (8.7–17.9) Sept This study 384 13 0.55 11.3a (6.9–21.5) May–Oct over 3 years Curtis and Vincent (2006) 58 10 0.57 n/a July–Nov Caldwell and Vincent (2012) 1674 6 0.53 n/a Year-round Correia (2015) 2042 n/a n/a 11.7 (7.1–16.6)b Year-round Vieira et al. (2014) MSP 19 0 0.62 11.8 (9.2–17.9) June This study RSP 31 n/a n/a n/a (4.2–7.3)c Year-round Verdiell‐Cubedo et al. (2006) TFR 25 0 0.36 13.0 (9.9–18.6) June, July, Aug This study 114 16 0.62 12.0 (8.1–16)b Year-round Louisy (2011) MIT 225 21 0.54 10.0 (7.0–14.0) June–Sept Gristina et al. (2015) KGR 14 7 0.46 13.0 (8.0–15.7) Sept This study CGR​ 13 0 0.53 11.2 (8.6–15.3) Sept This study VBU 60 2 0.68 6.4 (4.3–9.0) June This study TTR​ 272 n/a 0.50 8.3 (6.5–10.3)c Year-round Kasapoglu and Duzgunes (2014) NTR 139 n/a 0.42 n/a (5.7–9.0) n/a Başusta et al. (2014) WTR​ 135 n/a n/a 10.8 (6.4–13.2)cd n/a Filiz and Taskavak (2012) ATR​ 200 n/a 0.48 13.3 (10.0–16.5) Year-round Gurkan and Taskavak (2007) RGR​ 279 n/a 0.54 10.8 (7.8–22.5) Mar Kitsos et al. (2008) GTU​ 1773 n/a n/a 12.5 (6.3–17.6)ce Year-round Ben Amor et al. (2011) (b) H. hippocampus HUK 49 40 0.61 10.5 (5.6–19.8) Sept This study SUK 24 33 0.50 9.9 (7.1–16.8) April This study OUK 9 n/a n/a 9.4 (5.1–15.2) n/a Neil Garrick-Maidment Pers. Corr. BFR 16 0 0.50 10.2 (7.3–13.5) June This study AFR 13 13 0.54 10.6 (5.7–15.7) Sept, Oct, Nov This study GSP 9 n/a 0.34 n/a (11.8–17.1) Year-round Valladares et al. (2013) APO 9 n/a n/a n/a (4.5–13.7)c Year-round Veiga et al. (2009) PPO 6 0 0.33 8.3 (4.3–14.9) Sept This study RPO 44 0 0.60 8.7 (4.3–17.6) Sept, October This study 41 2 0.44 n/a (8.7–14.6) June–Sept Curtis and Vincent (2005) 18 28 0.38 n/a July–Nov Caldwell and Vincent (2012) 418 n/a n/a 8.3 (5.0–13.4)b Sept Vieira et al. (2014) 86 22 0.53 n/a Sept Correia (2015) MSP 23 0 0.52 8.5 (5.1–13.4) May, June This study GFR 21 0 0.61 8.5 (5.6–13.3) July This study GMA 5 0 0.60 9.1 (6.4–13.4) Aug This study MIT 16 6 n/a n/a June–Sept Gristina et al. (2015) RIT 46 n/a n/a 8.4 (5.7–10.3) March This study KGR 8 0 0.50 7.9 (5.7–11.1) Sept This study WTR​ 279 n/a n/a 8.4 (5.2–12.8)d n/a Filiz and Tasavak (2012)

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Table 2 (continued)

Site Total number Juvenile (%) Proportion of Ls (cm) Sampling period References females (min and max)

ATR​ 29 n/a 0.27 11.3 (7.9–14.0) Year-round Gurkan and Taskavak (2007) RGR​ 19 n/a 0.26 9.3 (6.9–10.4) Mar Kitsos et al. (2008) GTU​ 236 n/a n/a 10.9 (7.4–15.6) Year-round Ben Amor et al. (2011) LCN 19 0 0.52 8.4 (5.6–11.9) Nov This study GCN 165 20 0.58 10.2 (7.7–14.7) Year-round Otero-Ferrer et al. (2015a) SEN 40 n/a n/a 13.7 (10.8–18.1)d n/a This study

Number of seahorses samples, percentage of juveniles, sex ratio, standard length (Ls) and sampling period a At frst reproduction b Height c Total length d Dried specimens e ID as H. ramulosus

Table 3 Mean population Site Seahorses per diver hour Seahorses per ­m2 of transect References abundance include new and published data new for H. H. guttulatus H. hippocampus H. guttulatus H. hippocampus guttulatus and H. hippocampus BFR 1.565 1.130 – – This study GSP – – 0.007 – Valladares et al. (2011) TPO 3.000 0.006 – – This study PPO 6.000 0.980 – – This study RPO – – 0.073 0.007 Curtis and Vincent (2005) – – 0.004 0.001 Caldwell and Vincent (2012) – – 0.107 0.005 Correia (2015) SFR 0.980 0.001a – – This study – – 0.001–0.014b – Louisy (2011) GFR – 7.000 – – This study GMA – 0.190 – This study SIT – – – 0.006 Canese et al. (2007) MIT – – 0.018 > 0.001 Gristina et al. (2015) VIT – – 0.001 > 0.001 Franco et al. (2006) TSL – – 0–0.08 – Bonaca and Lipej (2005) KGR 1.070 1.000 0.020 0 This study – 0.004 – Issaris and Katsanevakis (2010) CGR​ 1.220 0.002 0.004 0 This study VBU 8.240c – 0.203 – This study LCN – 2.100 – 0.002 This study GCN – 1.760/0.840d – – Otero-Ferrer et al. (2015b)

a Only one seahorse seen but frst report of this species here b Range given not mean abundance c Abundance estimate was limited by underwater genetic sampling procedures d Calculated from 15 min dive transects

Formosa, Portugal, where observations covered many years, There is no signifcant diference in abundance of H. H. guttulatus was always found in greater abundance than H. guttulatus or H. hippocampus between sites where it hippocampus when the Ria Formosa was considered as one cohabits or not with its congener (H. guttulatus new data site, but in some locations within the Ria Formosa only one Mann–Whitney, W = 18, N = 10, P = 0.8; H. hippocampus of the two species was found (e.g. Curtis and Vincent 2005). all data Mann–Whitney U = 12, N = 7, P = 1). There was no

1 3 Marine Biology (2018) 165:19 Page 11 of 19 19 correlation between the density of the two seahorse species for either the data collected as ind. diver hour­ −1 (Spearman’s rho, rs = − 0.1, N = 5, P = 0.8) or in the Ria Formosa, Portu- gal, when abundance was measured as ind. ­m−2 (Spearman’s rho, rs = 0.2, N = 3, P = 0.8). Population structure

Each seahorse population had its own unique combination of characteristics, regards juvenile percentage and sex ratio (Table 2). In both species, the proportion of observed juve- niles varied widely, from 0 to 43% for H. guttulatus and from 0 to 40% for H. hippocampus. However, on average, this was about 17% across both species, and there appeared to be no efect of time of year. No H. guttulatus popula- tions were signifcantly male-biased, but TFR2 and VBU were both signifcantly female-biased (Chi Squared, TFR2: χ2 6.9, P < 0.01; VBU: χ2 8.1, P < 0.01), following Ben- jamini–Hochberg multiple comparison correction (false recovery rate 0.1). No H. hippocampus populations had a signifcant sex bias.

Abiotic parameters of seahorse habitat

There was a large variation in the environmental parameters of locations where seahorses were found. When all survey methods were considered, seahorses were found at depths ranging from 1 to 55 m, but when only dive surveys were included, seahorses were just found at 1–21 m. Most H. gut- Fig. 4 Holdfast substrate types utilized by H. guttulatus ( ) and H. tulatus (86%) were found at 2–5 m depth, from surveyed a hippocampus (b). Blues Organic, Browns Inorganic, Orange Artif- depths of 1–28 m. By contrast, just 19% of H. hippocam- cial, Green No holdfast (swimming) pus specimens were found in 2–5 m depth and at two sites (GMA&LCN) all were found much deeper (≥ 20 m). Neither water temperature nor visibility correlate with H. guttulatus Artifcial holdfasts were items such as tyres, fshing gear, abundance (Spearman’s rho, water temperature rs − 0.03, ropes, bricks and pier supports. Transect dives revealed that N = 8, P = 0.96, visibility rs − 0.50, N = 8, P = 0.20), or most seahorses were found in complex habitats on a sand/ with H. hippocampus abundance (Spearman’s rho, water silt substrate. These habitats included seagrass beds, sessile temperature rs − 0.38, N = 6, P = 0.34, visibility rs 0.04, invertebrates, algal species and artifcial structures (Table 4). N = 6, P = 0.94) when density is calculated as ind. diver Most sites that H. hippocampus inhabited were mixed ­hour−1. habitats of open sand or silt. These substrata often had added complexity due to the presence of sessile invertebrates, arti- Seahorse habitat and holdfast preference fcial structures or macroalgae. In addition, at six sites at least one species of seagrass was observed in the general New data from this study show most seahorses encountered vicinity of the seahorses. Hippocampus hippocampus was used holdfasts, with only 1% of H. guttulatus and 2% of H. most commonly found settled into depressions in the sedi- hippocampus seen while they were actively swimming. The ment. However artifcial structures were the most common seagrass Zostera marina was present at most sites (67%) holdfast used when they were present (19%) (Fig. 4). where H. guttulatus was observed. However seagrass beds Detailed descriptions of seahorse habitat and holdfast were not the dominant habitat (6.1 SE ± 4.2%) in any of the preference are lacking from many previous studies. However sites assessed with transect dives, although it was the most when reported, seagrass was present, but not dominant, at popular holdfast, accounting for just under half of H. guttu- most of the sites (86%). The seagrass observed was a mix of latus holdfasts (Fig. 4) (Table 1a). The second most popular genera: Cymodocea, Zostera and Posidonia. However, when holdfast type was artifcial structures (> 25% of seahorses). holdfast preference was recorded, macroalgae and artifcial

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Table 4 Survey dive data, habitat types observed from transects and quadrat surveys Site Species Transect habitat Quadrat habitat and percentage cover Dominant habitat Other habitats Bottom type % cover Flora/Fauna % cover Artifcial % cover

KGR H. hippocampus Sand Zostera/Cymo- Sand 41.2 Zostera 11.3 Brick 2.0 H. guttulatus docea mixed Gravel 38.0 Cymodocea 3.0 beds Stones 4.1 Urchins 0.2 Sea urchins Rock 0.2 CGR​ H. guttulatus Sand/Rock covered Gravel, urchin, Sand 51.0 Zostera 4.0 Brick 0.8 in algae sea cucum- Stones 33.6 Urchins 0.8 ber, gravel, Rock 7.4 Anemone 0.8 sponge Sponge 1.6 VBU H. guttulatus Sand Algae Sand 96.0 Mixed Chaetomor- 3.0 pha and Clad- 1.0 ophora spp. Hermit crab LCN H. hippocampus Sand Rock, rope, tyre Sand 98.0 Rock 0.5 Rope tyre 1.0 0.5 substrates were more often used by H. guttulatus, and inver- morphological characteristics was not possible as tissue tebrates used by H. hippocampus. Data were transformed was donated rather than whole specimens. This species into presence/absence due to the variety of methods and had previously been recorded in the south–eastern Medi- detail reported, but no individual factors could account for terranean Sea (Golani and Fine 2002; Gokoglu et al. 2004) seahorse presence (ANOSIM, seagrass R = 0.26, macroalgae and extant populations have been observed as far north R = − 0.01, sand R = 0.09, substrate complexity R = 0.18 as Turkey (Gokoglu et al. 2004). Hippocampus algiricus, and artifcial objects R = − 0.06). native to North–West African coasts (Czembor 2012), was observed in the Canary Islands (Otero-Ferrer et al. 2015b). By far the two most frequently seen species are H. gut- Discussion tulatus and H. hippocampus, which are native European species (Lourie et al. 2016). The present study provides the frst comprehensive review There was no genetic evidence within the samples tested of genetic and demographic information on European sea- for the presence of cryptic species or of substantial within- horses throughout their geographic range. It also provides species diferentiation (i.e. of sub-species level). This con- data on variability in morphology and habitat use between clusion could be drawn as all clades included reference and within H. guttulatus and H. hippocampus. Despite sam- sequences from known species and displayed no geographi- pling constraints, these baseline data provide useful informa- cally based or morphology-related structuring, despite tion for conservation assessments for these data poor species the high degree of intra-specifc morphological variation of conservation concern (Woodall 2012a, b). observed within and among sites (Otero-Ferrer et al. 2017). The only substantial within-species genetic diferentiation How many species of seahorse are there in European observed was that associated with haplotypes of H. hip- waters? pocampus from Senegal and some H. guttulatus haplotypes from the Black Sea (Fig. 2). However in both cases no Genetic data from 478 samples representing 18 loca- genetic and morphological correlation can be assumed, as tions collected in 10 countries revealed the presence of both Black Sea and Senegalese populations also possessed fve species of seahorse in NE Atlantic, Mediterranean the widely distributed common haplotypes of their respec- and Black Sea waters: H. algiricus, H. erectus, H. fus- tive species (Woodall et al. 2011, 2015). Levels of intra- cus, H. hippocampus, and H. guttulatus. One specimen specifc genetic variation in cytochrome b sequences for both from the Azores was identifed as H. erectus using genetic H. guttulatus and H. hippocampus (1.23 and 1.94%, respec- techniques, and subsequent morphological examination tively) were similar to values reported for other seahorse has confirmed this was the first observed occurrence species such as H. barbouri (1.72%, Lourie et al. 2005) and of this species in the eastern Atlantic Ocean (Woodall H. erectus (1.44%, Boehm et al. 2013). Intra-specifc varia- et al. 2009). Another species, H. fuscus, is an example of tion in the Control Region in H. guttulatus and H. hippocam- Lessepsian migration and was identifed from specimens pus (1.49 and 1.96%, respectively) likewise was within val- originating from northern Egypt, although congruence of ues given for other species such as H. abdominalis (2.23%,

1 3 Marine Biology (2018) 165:19 Page 13 of 19 19

Nickel and Cursons 2012), H. capensis (1.49%, Teske et al. Tagged H. hippocampus can shed cirri over time (JMR Cur- 2003) and H. ingens (2.10%, Saarman et al. 2010). tis, unpublished data) and in some locations in the UK, H. The lowest inter-specifc genetic variation among sea- hippocampus are never seen with cirri (Garrick-Maidment, horses has been reported between H. reidi and H. algiri- unpublished data).The general shape of cirri on H. guttulatus cus, consistent with these species having the most recent and H. hippocampus is often diferent, with cirri branching common ancestor (Teske et al. 2007a), whereas most other in a diferent manner (for example images see Figure S1). species pairwise comparisons show much greater genetic However these diferences are often unclear unless speci- divergence (e.g. 5.75% between H. erectus and H. patago- mens are compared simultaneously. As the genetic data nicus, Boehm et al. 2013). Although not conclusive, such clearly confrm the presence of just two native European levels of inter-specifc and intra-specifc genetic variation species, this current study can therefore confdently confrm across seahorse species suggest that the intra-specifc vari- cirri presence or absence is not a consistent or diagnosing ation observed across the entire geographical ranges of H. feature within species, concurring with Curtis (2006). guttulatus and H. hippocampus is consistent with these com- Sexual dimorphism, in the form of a shorter standard prising single undiferentiated species, and is also congruent length of males, was observed in H. guttulatus and H. hip- with the limited morphological diference seen. This is an pocampus. This was previously reported for H. guttulatus important conclusion, as previous studies based solely on (Curtis and Vincent 2006) and in many other seahorse spe- morphological data proposed new subspecies and species in cies (Foster and Vincent 2004), but the current study is the the Mediterranean Sea (Kuiter 2009) and Black Sea (Lourie frst to indicate that this is consistent for European seahorses et al. 1999b), that have subsequently been synonymised on across their entire geographic range. Sexual dimorphism is the basis of the genetic data (Woodall 2012a, b). This infor- generally a characteristic associated with polygamous spe- mation is crucial for surveying, assessing, monitoring, and cies, rather than monogamous ones like seahorses (Emlen managing the two focal species of this study, but could have and Oring 1977; Jones and Avise 2001), although H. guttu- wider ramifcations for seahorse taxonomy globally, where it latus is serially monogamous across breeding seasons (Naud is common for morphological characters alone to be used to et al. 2008). In seahorses the mating system is thought to describe species. Integrated taxonomy is recommended for be result from morphology, behaviour of mate competition, many species (Schlick-Steiner et al. 2010; Chen et al. 2011) and the energy required to produce eggs and brood them and conclusions from this study suggest this is particularly (Kvarnemo and Simmons 2013). important for seahorses where morphological diferentiation Adult H. guttulatus from the Black Sea were signif- can be challenging. cantly smaller than those from all other locations. This was observed for both new data (VBU) and published data Morphology in H. hippocampus and H. (TTR—Kasapoglu and Duzgunes 2014). In addition, H. hip- guttulatus is not consistent across their range pocampus from Senegal were larger than those from other or within populations sites. A signifcant size diference of seahorses from difer- ent populations has not been observed previously, however Previous studies have relied on a subset of morphological a large range of sizes have been reported for both H. hip- characters that are distinctive in seahorses (Lourie et al. pocampus and H. guttulatus (Table 2), morphological vari- 1999b) to determine taxonomy. Data on two commonly used ation has been seen across Macaronesia and W. Africa in characteristics (presence/absence of cirri, standard length) H. hippocampus (Otero-Ferrer et al. 2017), and phenotypic show a difering proportion of individuals with cirri in each plasticity is recognised in other seahorse species (Teske population of both species, and results indicate that the pres- et al. 2007b). In some species of pipefsh, which are in the ence and number of cirri are unreliable characters for Euro- same family as seahorses, lengths are signifcantly diferent pean seahorse species identifcation. This result is congruent between populations (e.g. Syngnathus foridae, Mobley and with the few other studies that recorded this morphological Jones 2009; Syngnathus typhle, Rispoli and Wilson 2008), character (Curtis and Vincent 2006; Curtis 2006; Louisy and another pipefsh species, Syngnathus abaster, appears 2011; Tiralongo and Baldacconi 2014; Otero-Ferrer et al. to be morphologically divergent across diferent locations 2015a). Cirri presence was more likely on larger H. guttula- (Cakic et al. 2002; Veiga et al. 2009; Ben Alaya et al. 2011). tus supporting fndings by Curtis (2006), however this is not Based on mtDNA data, these studies suggest the morpho- always the case (Garrick-Maidment pers. comm.), suggest- logical diferences between populations are probably linked ing that the conditions required to exhibit this character are to genetic diferentiation in S. abaster, whereas ecological highly complex. In H. hippocampus however, females were factors are a more likely cause for the morphological vari- more likely to have cirri. This was congruent with a study ation observed in other pipefsh species as no genetic cor- in the Canary Islands by Otero-Ferrer et al. (2015a) which relation is seen (Mobley and Jones 2009). In the present reported females were more likely to have cirri than males. study the size of both focal species was diferent across sites.

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Additional studies are required to elucidate which location- ­m−2) employed in the current study showed that across their specifc factors correlate with the observed size diferences range H. guttulatus abundance was greater than that of H. in seahorses. Despite the apparent trend for seahorses to be hippocampus. The abundance of H. guttulatus was always larger in the most northerly locations (a proxy of seasonal greater than that of H. hippocampus in locations where they variation in temperatures), these fndings are not signif- co-occurred. Other studies have shown this pattern over lim- cant and therefore neither European seahorse species fol- ited geographic areas (Curtis and Vincent 2005; Caldwell low Bergmann’s rule. This rule states larger individuals are and Vincent 2012; Gristina et al. 2015). Just one other study found in colder environments, and smaller ones in warmer reports percentage abundance of co-occurring seahorse spe- ones. This nonconformity could be a sampling artefact, but cies, which revealed the same composition of species and the may also refect that an organism’s size is infuenced by a same most abundant species (Murugan et al. 2008). Counter complex range of ecological and evolutionary processes to this in pipefsh the species of greatest abundance appears (Berke et al. 2013), and seahorse survival requirements are to be related to season (Ripley and Foran 2006) and micro- known to be complex. It is therefore unsurprising that they habitat preference (Malavasi et al. 2007). show morphological variation across their geographic range The female-biased sex ratio of H. guttulatus in two of as an adaptation to diferent local conditions, similar to that the sites, in the Black Sea and southern France (VBU in observed in their confamilial Syngnathus leptorhynchus this study and TFR2 in Louisy 2011) is unexpected as serial (Wilson 2009). monogamy reported for H. guttulatus in an ex situ trial and over 2 years in the wild (Naud et al. 2008) and over 4 years Population demographics in the wild at a UK site (Garrick Maidment unpublished data) predicts an equal sex ratio. An independent study (new Seahorse density was generally low, but patchy and highly data in the present study) of site TFR reported an equal sex variable. New abundance estimates were within values ratio, which might suggest the female bias of the Louisy given in other studies of both species (Table 1), but few (2011) may be an anomaly and additional data should be report density using ind. diver ­hour−1, which comprises the collected to investigate this further. Seasonal changes in the majority of new data in this study. No direct comparison sex ratio have been reported for H. zostera (Strawn 1958), a between ind. m­ −2 and ind. diver hour­ −1 was possible. Safety female biased population was documented in H. abdominalis considerations during diving, such as depth, water clarity, (Martin-Smith and Vincent 2005), and an equal sex ratio was current fow rate and boat trafc limited the possible search observed for H. comes (Perante 2002), suggesting a variety area within known seahorse sites (Curtis and Vincent 2005; of sex ratios can be observed across seahorse species. All H. Curtis et al. 2017). All new study sites were chosen because hippocampus populations in the present study had an equal seahorses had previously been observed at them, therefore sex ratio, with most individuals found as male/female pairs abundance presented is artificially inflated. The choice (pers. obs.), although seasonal changes have been indicated of search method has also been shown to infuence abun- in one study (Otero-Ferrer et al. 2015a). This interesting dance recorded, which in most cases will have also infated diference between species should be investigated further abundance reported (Correia et al. 2016). However, mean to determine if this phenomenon is a possible characteristic density for H. hippocampus from new data presented here for niche partitioning in these species, especially as mat- are within values extrapolated from previous studies of H. ing behaviour studies have not yet been conducted for H. hippocampus (Otero-Ferrer et al. 2015a). Data from both hippocampus. species combined (Gofredo et al. 2004) suggests that this The number of juveniles seen in surveyed populations method could be useful for surveys and comparisons with is fewer than adults. However, juveniles could be observed distance transect measures, should be a priority. Densities less frequently that adults due to the sampling method and reported in previous studies are from transects or focal grids. regime or as a result of an ontogenetic habitat shift. Most The latter are often chosen to encompass areas of high sea- studies of other seahorse species (reviewed in Foster and horse density (e.g. Bell et al. 2003) and therefore seahorse Vincent 2004), including H. guttulatus (Correia 2015; Gris- densities from focal studies would be artifcially higher com- tina et al. 2017), also documented low proportions juveniles, pared to randomly placed transects. The seahorse densities however a high proportion has been found in some popu- given per area surveyed in the present study were generally lations of H. capensis (Lockyear et al. 2006). There is a similar to those previously reported in these species (e.g. precedent for ontogeny in seahorses (H. comes, Morgan Gristina et al. 2015), but greater than those reported for other and Vincent 2007; H. whitei, Harasti et al. 2014). Further seahorse species (Foster and Vincent 2004). This may be research is required to understand this aspect of behaviour in an artefact of the sampling protocol as mentioned above, European seahorses, although has been observed in H. hip- a species-specifc characteristic or peculiarity of sample pocampus in the Canary Islands (Otero-Ferrer et al. 2015a). location. Both abundance measures (ind. diver ­hour−1, ind. This is especially important as best practice dictates that the

1 3 Marine Biology (2018) 165:19 Page 15 of 19 19 efective management and conservation of species needs to and environment parameters alone is challenging. This is an address all life stages (Gerber and Heppell 2004). important consideration for environmental assessments that are made before potentially damaging activities (e.g. coastal Can seahorse population location be predicted construction). The requirements of such assessments difer by environmental parameters? across states, however habitat is often used as a precursor to determine which species (such as seahorses) could be at risk. In this study, no individual environmental parameters Our fndings suggest that this strategy would not be suit- could defne the presence of seahorses, species abundance, able for determining potential impacts on H. hippocampus or determine which species was present. However survey and H. guttulatus. Furthermore, as seahorse conservation locations were not picked at random, with only locations eforts are currently associated with seagrass conservation where seahorses were already known to be present being (e.g. Heritage Lottery Fund 2014), much of the variation in studied, which may have limited our ability to detect envi- European seahorse habitat may be missed if seagrass beds ronmental parameters that are unsuitable for seahorses. In alone are conserved, despite this habitat being important for order to model where seahorses may occur, it is important to many other species (McCloskey and Unsworth 2015). identify how diferent locations and habitats fulfl the needs of seahorses. These factors could include environmental Important new insights and future research parameters under extreme events like storms and extended suggestions to enable appropriate conservation periods of heat (Cohen et al. 2017). Correlation of H. gut- measures tulatus abundance and temperature has been reported for multiple populations within the Ria Formosa (Correia 2015), This study provides the frst synthesis of data on habitat, although neither visibility nor temperature appeared to cor- population demography, morphology and genetics of the two relate with seahorse sightings in a UK site (The Seahorse native European seahorse species H. guttulatus and H. hip- Trust 2014). pocampus from across their geographic range. We report the When only new data were analysed, H. guttulatus was large variety of habitats in which these fsh are found, failed most commonly seen in complex habitats and H. hippocam- to identify one simple parameter that predicts the presence pus in simpler ones; supporting previous location-specifc or abundance of these seahorses, but note that seagrass is studies (Curtis and Vincent 2005; Canese et al. 2007; Cor- not always associated with either species. Data show that reia 2015; Garrick-Maidment 2011; Gristina et al. 2015; the morphology of specimens should be carefully consid- Otero-Ferrer et al. 2015a). Niche partitioning was also ered together with genetic data, in an integrated approach, in observed in sympatric pipefsh (Kendrick and Hyndes 2003; order to assign species identifcations (Feulner et al. 2007); Malavasi et al. 2007 and in the pygmy seahorses H. denise such accurate integrated identifcation is vital in order to and H. bargibanti Smith et al. 2012). There is inconclusive allow international legal mechanisms and international evidence of the importance of Zostera marina as a required agreements such as the Convention on International Trade or preferred habitat for H. guttulatus, as although it did not in Endangered Species (CITES 2015) to work efectively. always co-occur with seahorse populations, when present it Emerging techniques such as eDNA screening could was most often used by H. guttulatus as a holdfast. Although be applied to locate hitherto unknown populations, which Z. marina itself could be important for H. guttulatus, it is would be valuable for understanding the distribution and more likely to be the food availability as infauna and epi- ecology of these species. The diferences in abundance fauna associated with the seagrass (Bostrom and Bonsdorf observed between the two species suggest diferent condi- 1997) that is driving habitat preference, as is the case with tions are required for these species to thrive, but these exact pipefsh (Ryer and Orth 1987). Individuals of both H. gut- parameters are yet to be determined. Niche partitioning is tulatus and H. hippocampus were observed using artifcial expected in congeneric species, and further observations to objects as holdfasts. The use of artifcial holdfasts is seen determine any diferences in prey items (Kitsos et al. 2008), in many seahorse species (Rosa et al. 2007; Clynick 2008; morphology and behaviour would be an interesting contribu- Faleiro et al. 2008), and could be an important factor in man- tion to determine how management measures diferentially agement measures as they could provide refuge for seahorse impact the two species. prey items or function as seahorse aggregation devices (Cor- As both H. guttulatus and H. hippocampus are currently reia et al. 2013, 2015), but this apparent behavioural pref- classifed as Data Defcient (IUCN 2015), any range-wide erence may be an observer artefact because they are more conservation measures should also encompass long-term easily seen on this type of object. monitoring so that the threat status of these iconic fsh can The apparent wide range of habitats means that predict- be reassessed. Especially as population trends are unknown ing the likelihood of these species’ presence from habitat in many locations. Applying the precautionary principal,

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Aquat Conserv 22:427–435. https://doi.org/10.1002/ Compliance with ethical standards aqc.2238 Canese S, Giusti M, Salvati E, Angiolillo M, Cardinali A, Fabroni F, Funding Celia-Magno M, Greci S (2007) Preliminary note of the presence Funding for this project was provided by Chocolaterie and density of Hippocampus hippocampus in the Soverato Bay, Guylian and a Natural Environment Research Council Industrial Case Calabria Ionica. Biol Mar Mediterr 14:340–341 studentship (NER/S/C/2005/13461) to LCW. Support for FOF was Casey S, Hall H, Stanley HF, Vincent AC (2004) The origin and provided by the European Commission (ASSEMBLE project, Grant evolution of seahorses (genus Hippocampus): a phylogenetic agreement no. 227799). study using the cytochrome b gene of mitochondrial DNA. Mol Phylogenet Evol 30:261–272. https://doi.org/10.1016/j. Conflict of interest The authors declare that they no conficts of inter- ympev.2003.08.018 est. Chen J, Li Q, Kong L, Yu H (2011) How DNA barcodes comple- ment taxonomy and explore species diversity: the case study of Ethical approval All applicable international, national, and institutional a poorly understood marine fauna. PLoS One 6:e21326. https:// guidelines for the care and use of animals were followed. doi.org/10.1371/journal.pone.0021326 Cisneros-Montemayor AM, West K, Boiro IS, Vincent ACJ (2016) An assessment of West African seahorses in fsheries catch and Open Access This article is distributed under the terms of the Creative trade. J Fish Biol 88:751–759. https://doi.org/10.1111/jfb.12818 Commons Attribution 4.0 International License (http://creativecom- CITES (2015) What is CITES http://www.cites.org/eng.disc/what.php. mons.org/licenses/by/4.0/), which permits unrestricted use, distribu- Accessed 13 Nov 2015 tion, and reproduction in any medium, provided you give appropriate Clynick BG (2008) Characteristics of an urban fsh assemblage: dis- credit to the original author(s) and the source, provide a link to the tribution of fsh associated with coastal marinas. Mar Environ Creative Commons license, and indicate if changes were made. Res 65:18–33. https://doi.org/10.1016/j.marenvres.2007.07.005 Cohen FPA, Valenti WC, Planas M, Calado R (2017) Seahorse aqua- culture, biology and conservation: knowledge gaps and research

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